Non-invasive imaging of an inflammatory response or of an immune response against a tumor remains a highly desirable yet challenging goal. A combination of leukocyte-specific single-domain antibodies (VHHs) and a sortase-based enzymatic conjugation reaction will be used for the creation of immunodiagnostics suitable for positron emission tomography (PET) to track immune response and to image inflammation. The enzymatic modification platform readily allows the site-specific and rapid installation of 18F, 64Cu or 68Ga onto VHHs that will be chosen from a panel recognizing human and mouse antigens, including Class II MHC, CD3, CD4, CD8, CTLA4, PD-1 and PD-L1. These studies will involve optimization of labeling efficiency and pharmacokinetic analysis by PET imaging of lead candidates. We shall analyze the distribution of leukocytes in normal mice, in mice in which inflammation is induced, and in xenografted and syngeneic tumor models. Our preliminary data show the generation of a site-specifically 18F-labeled single domain antibody specific for murine Class II MHC products, 18F-VHH7. We used 18F-VHH7 to perform positron emission tomography (PET) in mice, using wild type, MHC II-/- and NOD-SCID mice xenografted with a human melanoma cell line. Not only is 18F-VHH7 rapidly cleared from the circulation, (t 1/2<20 min), it also reveals secondary lymphoid organs with remarkable specificity. Moreover, Class II MHC+ cells associated with the melanoma xenograft were clearly visualized with 18F-VHH7, setting the stage for early non-invasive detection of inflammatory cells and lymphocytes as an indicator of disease. We propose the following specific aims: Aim 1. Develop a labeling strategy to generate 18F-labeled single domain alpaca-derived antibodies (VHHs) against mouse CD3??, CD11b and Class II MHC for use in PET imaging. We will also develop a method for site-specific protein labeling with radiometals such as 68Ga or 64Cu, using NOTA as chelating agent. Aim 2. Isolate single domain antibodies from a phage display library obtained from an alpaca immunized with recombinant mouse CTLA4, CD4, CD8, PD-1 and PD-L1 in a sortase-ready format to enable labeling with 18F, 68Ga or 64Cu. Aim 3. Using the tools developed in Aims 1 and 2 perform PET imaging to explore the distribution of leukocytes in normal mice, in animals in which inflammation is induced, and in mice with xenografted and syngeneic tumors. The successful completion of these aims will provide the necessary foundation for translation of this approach to a more clinical setting.